Cystic fibrosis (CF) is the most common lethal inherited disease in the caucasian population. Despite recent major advances in the isolation and characterization of the CF gene, the structure and function of the wild type protein product, named the CF transmembrane conductance regulator (CFTR), has not been defined. CFTR is predicted to be an integral membrane glycoprotein. The specific aims of this proposal are to test the hypotheses that mutations in the CF gene result in the altered glycosylation of CFTR, and that this is associated with altered function and/or subcellular localization of CFTR as well as with altered glycosylation of other glycoproteins in airway epithelial cells. Altered glycosylation of glycoproteins in CF is well established in other systems. CFTR will be isolated from airway epithelial cells by immunoaffinity and biochemical techniques. Polyclonal antibodies were generated to peptides from two distinct domains of CFTR. These antibodies identify a protein, Mr 170,000, the predicted molecular size of CFTR, in western blot analysis of airway epithelial cells. After confirming the authenticity, CFTR will be biochemically characterized with a particular emphasis on glycosylation, defining the oligosaccharides by lectin affinity and 500-MHz H-NMR spectroscopy. The subcellular localization will be defined by immunohistochemistry, cell fractionation, pulse/chase labelling, and inhibitors of glycosylation. In experiments which may perturbate function, correlation will be made with chloride conductance to determine if this CF phenotype is altered. Bovine CFTR cDNA is highly homologous to the human. A full length, bovine CFTR cDNA will be used to synthesize RNA for microinjection into Xenopus oocytes to further define oligosaccharide processing and localization of CFTR utilizing site-directed mutagenesis. The studies proposed for the airway cells will provide direct information on the glycosylation and localization of CFTR when both wild type and the most common CF mutations are examined in an epithelial cell environment. The experiments in oocytes will corroborate and extend these studies by addressing both common and unusual mutations of CFTR in a different cellular environment. The long range goals of this project are to define the pathophysiology of CF. These goals are based on the conviction that a thorough understanding of CF at the molecular and cellular level is a necessary prerequisite to designing effective strategies for the rational therapy of CF.